Alzheimer's disease (AD) is an increasingly prevalent form of neurodegeneration that accounts for approximately 50%-60% of the overall cases of dementia among people over 65 years of age. Death of pyramidal neurons and loss of neuronal synapses in brains regions associated with higher mental functions results in the typical symptoms, characterized by gross and progressive impairment of cognitive function. Neuropathologically, the major hallmarks of AD are the presence of two characteristic lesions: the amyloid senile plaque and neurofibrillary tangle (NFT). While the plaque is deposited extraneuronally, the tangle is observed intraneuronally in the post-mortem brain. One of the major components of the amyloid plaque core is the pathologically deposited small amyloid-beta-peptide (Aβ), which is cleaved by secretases from amyloid precursor protein (APP). Aβ, a self-aggregating peptide of 39-43 residues (MW˜4 kDa), is synthesized as part of the larger APP (110-120 kDa). APP is a type I integral membrane glycoprotein with a large N-terminal extracellular domain, a single transmembrane domain and a short cytoplasmic tail. The Aβ region spans portions of the extracellular and transmembrane domains of APP. The most common hypothesis for the participation of APP in neuronal cell death in AD is the amyloid hypothesis. This hypothesis postulates that plaque amyloid depositions or partially aggregated soluble AB trigger a neurotoxic cascade, thereby causing neurodegeneration similar to AD pathology.
Insulin-like growth factor I (IGF-I) is a circulating hormone structurally related to insulin. IGF-I was traditionally considered the major mediator of the actions of growth hormone on peripheral tissues IGF-I consists of 70 amino acids and is also named somatomedin C and defined by SwissProt No. P01343. Use, activity and production are mentioned in, e.g., EP 0 123 228; EP 0 128 733; U.S. Pat. No. 5,861,373; U.S. Pat. No. 5,714,460; EP 0 597 033; WO 02/32449; WO 93/02695.
The regulation of IGF-I function is quite complex. In the circulation, only 0.2% of IGF-I exists in the free form whereas the majority is bound to IGF-binding proteins (IGFBP's), which have very high affinities to IGF's and modulate IGF-I function. The factor can be locally liberated by mechanisms releasing IGF-I such as proteolysis of IGFBPs by proteases.
IGF-I plays a paracrine role in the developing and mature brain, and in vitro studies indicate that IGF-I is a potent non-selective trophic agent for several types of neurons in the CNS.
Reduction of brain and serum levels of free IGF-I has been related to the pathogenesis of sporadic and familial forms of AD. Furthermore, IGF-I protects neurons against Aβ-induced neurotoxicity. Peripherally administered IGF-I is capable of reducing brain Aβ levels in rats and mice and that in a transgenic AD mouse model prolonged IGF-I treatment significantly reduced brain amyloid plaque load. These data strongly support the idea that IGF-I is able to reduce brain Aβ levels and plaque-associated brain dementia by clearing Aβ from the brain.
Covalent modification of proteins with poly(ethylene glycol) (PEG) has proven to be a useful method to extend the circulating half-lives of proteins in the body. Other advantages of PEGylation are an increase of solubility and a decrease in protein immunogenicity. A common method for the PEGylation of proteins is the use of poly(ethylene glycol) activated with amino-reactive reagents like N-hydroxysuccinimide (NHS). With such reagents poly(ethylene glycol) is attached to the proteins at free primary amino groups such as the N-terminal α-amino group and the ε-amino groups of lysine residues. However, a major limitation of this approach is that proteins typically contain a considerable amount of lysine residues and therefore the poly(ethylene glycol) groups are attached to the protein in a non-specific manner at all of the free ε-amino groups, resulting in a heterologous product mixture of random PEGylated proteins. Therefore, many NHS-PEGylated proteins are unsuitable for commercial use because of low specific activity. Inactivation results from covalent modification of one or more lysine residues or the N-terminal amino residue required for biological activity or from covalent attachment of the poly(ethylene glycol) residues near or at the active site of the protein.
WO 94/12219 and WO 95/32003 claim polyethylene glycol conjugates comprising PEG and IGF or a cysteine-mutated IGF, where the PEG is attached to said mutein at a free cysteine in the N-terminal region of the mutein. WO 2004/60300 describes N-terminally PEGylated IGF-I.